The long-term goal of this research is to identify molecular targets that could be used for new mental health therapeutics, particularly for affective disorders that differ between the sexes. We will approach this goal by identifying synaptic proteins that are acutely regulated by estradiol (E2) and/or agonists selective for the form of the estrogen receptor (ER in the hippocampus of adult male and female rats. The rationale for this approach is as follows: First, the hippocampus is a brain region implicated in affective behaviors and E2 or ER agonists infused directly into the hippocampus acutely decrease anxiety-related behaviors in females but not in males. Second, there is strong evidence that E2 is produced locally as a neurosteroid in the hippocampus of both sexes, providing a physiological source of E2 that could acutely modulate affective behavior in vivo. Third, we have found that E2 and ER agonists have differential effects on synaptic transmission in the hippocampus of males and females with the same time course as their effects on anxietylike behavior in females. These findings support the idea that sex differences in acute ER-dependent synaptic modulation in the hippocampus could contribute to sex differences in anxiety-like behaviors. An essential step in moving these behavioral and electrophysiological studies toward future, focused efforts to develop novel mental health therapeutics is to identify specific molecular mechanisms that act downstream of acute E2-ER signaling to modulate hippocampal synaptic physiology and behavior. Here, we propose to use two unbiased and complementary proteomics approaches to identify synaptic proteins that are acutely regulated by E2 and/or ER agonists in the hippocampus of male and female rats. In Aim 1, we will use two dimensional difference in gel electrophoresis followed by mass spectrometry to identify synaptic proteins that are differentially regulated by acute ER activation in the hippocampus of males vs. females. In Aim 2, we will use strong cation exchange chromatography and titanium dioxide enrichment of phosphopeptides followed by mass spectrometry to identify synaptic proteins that are differentially phosphorylated by acute ER activation in the hippocampus of males vs. females. Upon completion of this project, we will have a broad and unbiased profile of synaptic proteins the levels and/or phosphorylation of which differ between males and females, and/or that are differentially regulated by acute ER activation in the hippocampus of males vs. females. Because we will use treatments and timing that are the same as in ongoing behavioral and electrophysiological studies, we will be able to relate specific molecular changes that we discover with proteomics to sex differences in regulation of anxiety-like behaviors and of synaptic function in the hippocampus. Discovery of sex-specific mechanisms of synaptic protein regulation in the hippocampus could identify biomarkers for susceptibility to affective disorders and ultimately lead to sex-specific treatments.